Jiraporn Ousingsawat

Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport.

Molecular identification of the Ca2+-dependent chloride channel TMEM16A (ANO1) provided a fundamental step in understanding Ca2+-dependent Cl− secretion in epithelia. TMEM16A is an intrinsic constituent of Ca2+-dependent Cl− channels in cultured epithelia and may control salivary output, but its physiological role in native epithelial tissues remains largely obscure. Here, we demonstrate that Cl− secretion in native epithelia activated by Ca2+-dependent agonists is missing in mice lacking expression of TMEM16A. Ca2+-dependent Cl− transport was missing or largely reduced in isolated tracheal and colonic epithelia, as well as hepatocytes and acinar cells from pancreatic and submandibular glands of TMEM16A−/− animals. Measurement of particle transport on the surface of tracheas ex vivo indicated largely reduced mucociliary clearance in TMEM16A−/− mice. These results clearly demonstrate the broad physiological role of TMEM16A−/− for Ca2+-dependent Cl− secretion and provide the basis for novel treatments in cystic fibrosis, infectious diarrhea, and Sjöegren syndrome.

TMEM16 proteins produce volume regulated chloride currents that are reduced in mice lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca2+-activated Cl− channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y2 receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca2+ and a Ca2+-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl− conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of ICl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl− channels and may also have a function during proliferation and apoptotic cell death.

Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A

TMEM16A (anoctamin 1, Ano1), a member of a family of 10 homologous proteins, has been shown to form an essential component of Ca2+‐activated CU channels. TMEM16A‐null mice exhibit severe defects in epithelial transport along with tracheomalacia and death within 1 mo after birth. Despite its outstanding physiological significance, the mechanisms for activation of TMEM16A remain obscure. TMEM16A is activated on increase in intracellular Ca2+, but it is unclear whether Ca2+ binds directly to the channel or whether additional components are required. We demonstrate that TMEM16A is strictly membrane localized and requires cytoskeletal interactions to be fully activated. Despite the need for cytosolic ATP for full activation, phosphorylation by protein kinases is not required. In contrast, the Ca2+ binding protein calmodulin appears indispensable and interacts physically with TMEM16A. Openers of small‐ and intermediate‐conductance Ca2+‐activated potassium channels known to interact with calmodulin, such as 1‐EBIO, DCEBIO, or riluzole, also activated TMEM16A. These results reinforce the use of these compounds for activation of electrolyte secretion in diseases such as cystic fibrosis.—Tian, Y., Kongsuphol, P., Hug, M., Ousingsawat, J., Witzgall, R., Schreiber, R., Kunzelmann, K. Calmodulin‐dependent activation of the epithelial calcium‐dependent chloride channel TMEM16A. FASEB J. 25, 1058–1068 (2011). www.fasebj.org

Bestrophin and TMEM16—Ca2+ activated Cl− channels with different functions

In the past, a number of candidates have been proposed to form Ca(2+) activated Cl(-) currents, but it is only recently that two families of proteins, the bestrophins and the TMEM16-proteins, recapitulate reliably the properties of Ca(2+) activated Cl(-) currents. Bestrophin 1 is strongly expressed in the retinal pigment epithelium, but also at lower levels in other cell types. Bestrophin 1 may form Ca(2+) activated chloride channels and, at the same time, affect intracellular Ca(2+) signaling. In epithelial cells, bestrophin 1 probably controls receptor mediated Ca(2+) signaling. It may do so by facilitating Ca(2+) release from the endoplasmic reticulum, thereby indirectly activating membrane localized Ca(2+)-dependent Cl(-) channels. In contrast to bestrophin 1, the Ca(2+) activated Cl(-) channel TMEM16A (anoctamin 1, ANO1) shows most of the biophysical and pharmacological properties that have been attributed to Ca(2+)-dependent Cl(-) channels in various tissues. TMEM16A is broadly expressed in both mouse and human tissues and is of particular importance in epithelial cells. Thus exocrine gland secretion as well as electrolyte transport by both respiratory and intestinal epithelia requires TMEM16A. Because of its role for Ca(2+)-dependent Cl(-) secretion in human airways, it is likely to become a prime target for the therapy of cystic fibrosis lung disease, caused by defective cAMP-dependent Cl(-) secretion. It will be very exciting to learn, how TMEM16A and other TMEM16-proteins are activated upon increase in intracellular Ca(2+), and whether the other nine members of the TMEM16 family also form Cl(-) channels with properties similar to TMEM16A.

Expression of Voltage-Gated Potassium Channels in Human and Mouse Colonic Carcinoma

Purpose: Voltage-gated Kv potassium channels, like ether a go-go (EAG) channels, have been recognized for their oncogenic potential in breast cancer and other malignant tumors. Experimental Design: We examined the molecular and functional expression of Kv channels in human colonic cancers and colon of mice treated with the chemical carcinogens dimethylhydrazine and N-methyl-N-nitrosourea. The data were compared with results from control mice and animals with chemically induced DSS colitis. Results: Electrogenic salt transport by amiloride-sensitive Na+ channels and cyclic AMP–activated cystic fibrosis transmembrane conductance regulator Cl− channels were attenuated during tumor development and colitis, whereas Ca2+-dependent transport remained unchanged. Kv channels, in particular Eag-1, were enhanced during carcinogenesis. Multiplex reverse transcription-PCR showed increased mRNA expression for Kv1.3, Kv1.5, Kv3.1, and members of the Eag channel family, after dimethylhydrazine and N-methyl-N-nitrosourea treatment. Eag-1 protein was detected in the malignant mouse colon and human colonic cancers. Genomic amplification of Eag-1 was found in 3.4% of all human colorectal adenocarcinoma and was an independent marker of adverse prognosis. Conclusions: The study predicts an oncogenic role of Kv and Eag channels for the development of colonic cancer. These channels may represent an important target for a novel pharmacotherapy of colonic cancer.

Voltage-gated K+ channels support proliferation of colonic carcinoma cells

Plasma membrane potassium (K+) channels are required for cell proliferation. Evidence is growing that K+ channels play a central role in the development and growth of human cancer. Here we examine the contribution and the mechanism by which K+ channels control proliferation of T84 human colonic carcinoma cells. Numerous K+ channels are expressed in T84 cells, but only voltage‐gated K+ (Kv) channels influenced proliferation. A number of Kv channel inhibitors reduced DNA synthesis and cell number, without exerting apoptotic or toxic effects. Expression of several Kv channels, such as EagI, Kv 3.4 and Kv 1.5, was detected in patch clamp experiments and in fluorescence‐based assays using a voltage sensitive dye. The contribution of EagI channels to proliferation was confirmed by siRNA, which abolished EagI activity and inhibited cell growth. Inhibition of Kv channels did not interfere with the ability of T84 cells to regulate their cell vol, but it restricted intracellular pH regulation. In addition, inhibitors of Kv channels, as well as siRNA for EagI, attenuated intracellular Ca2+ signaling. The data suggest that Kv channels control proliferation of colonic cancer cells by affecting intracellular pH and Ca2+ signaling. Spitzner, M., Ousingsawat, J., Scheidt, K., Kunzelmann, K., Schreiber, R. Voltage‐gated K+ channels support proliferation of colonic carcinoma cells. FASEB J. 21, 35–44 (2007)

Disruption of the K+ Channel β-Subunit KCNE3 Reveals an Important Role in Intestinal and Tracheal Cl− Transport

The KCNE3 β-subunit constitutively opens outwardly rectifying KCNQ1 (Kv7.1) K+ channels by abolishing their voltage-dependent gating. The resulting KCNQ1/KCNE3 heteromers display enhanced sensitivity to K+ channel inhibitors like chromanol 293B. KCNE3 was also suggested to modify biophysical properties of several other K+ channels, and a mutation in KCNE3 was proposed to underlie forms of human periodic paralysis. To investigate physiological roles of KCNE3, we now disrupted its gene in mice. kcne3−/− mice were viable and fertile and displayed neither periodic paralysis nor other obvious skeletal muscle abnormalities. KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they might facilitate transepithelial Cl− secretion through basolateral recycling of K+ ions and by increasing the electrochemical driving force for apical Cl− exit. Indeed, cAMP-stimulated electrogenic Cl− secretion across tracheal and intestinal epithelia was drastically reduced in kcne3−/− mice. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3−/− mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. Further, these results suggest KCNE3 as a potential modifier gene in cystic fibrosis.

Molecular functions of anoctamin 6 (TMEM16F): a chloride channel, cation channel, or phospholipid scramblase?

Anoctamin 6 (Ano6; TMEM16F gene) is a ubiquitous protein; the expression of which is defective in patients with Scott syndrome, an inherited bleeding disorder based on defective scrambling of plasma membrane phospholipids. For Ano6, quite diverse functions have been described: (1) it can form an outwardly rectifying, Ca2+-dependent and a volume-regulated Cl− channel; (2) it was claimed to be a Ca2+-regulated nonselective cation channel permeable for Ca2+; (3) it was shown to be essential for Ca2+-mediated scrambling of membrane phospholipids; and (4) it can regulate cell blebbing and microparticle shedding. Deficiency of Ano6 in blood cells from Scott patients or Ano6 null mice appears to affect all of these cell responses. Furthermore, Ano6 deficiency in mice impairs the mineralization of osteoblasts, resulting in reduced skeletal development. These diverse results have been obtained under different experimental conditions, which may explain some of the contradictions. This review therefore aims to summarize the currently available information on the diverse roles of Ano6 and tries to clear up some of the existing controversies.

Rotavirus toxin NSP4 induces diarrhea by activation of TMEM16A and inhibition of Na+ absorption

Rotavirus infection is the most frequent cause for severe diarrhea in infants, killing more than 600,000 every year. The nonstructural protein NSP4 acts as a rotavirus enterotoxin, inducing secretory diarrhea without any structural organ damage. Electrolyte transport was assessed in the colonic epithelium from pups and adult mice using Ussing chamber recordings. Western blots and immunocytochemistry was performed in intestinal tissues from wild-type and TMEM16A knockout mice. Ion channel currents were recorded using patch clamp techniques. We show that the synthetic NSP4114–135 peptide uses multiple pro-secretory pathways to induce diarrhea, by activating the recently identified Ca2+-activated Cl− channel TMEM16A, and by inhibiting Na+ absorption by the epithelial Na+ channel ENaC and the Na+/glucose cotransporter SGLT1. Activation of secretion and inhibition of Na+ absorption by NSP4114–135, respectively, could be potently suppressed by wheat germ agglutinin which probably competes with NSP4114–135 for binding to an unknown glycolipid receptor. The present paper gives a clue as to mechanisms of rotavirus-induced diarrhea and suggests wheat germ agglutinin as a simple and effective therapy.

AMPK controls epithelial Na+ channels through Nedd4-2 and causes an epithelial phenotype when mutated

The metabolic sensor adenosine-monophosphate-activated kinase (AMPK) detects the cellular energy status and adjusts metabolic activity according to the cytosolic AMP to ATP ratio. Na+ absorption by epithelial Na+ channels (ENaC) is a highly energy-consuming process that is inhibited by AMPK. We show that the catalytic subunit α1 of AMPK inhibits ENaC in epithelial tissues from airways, kidney, and colon and that AMPK regulation of ENaC is absent in AMPKα1−/− mice. These mice demonstrate enhanced electrogenic Na+ absorption that leads to subtle changes in intestinal and renal function and may also affect Na+ absorption and mucociliary clearance in the airways. We demonstrate that AMPK uses the ubiquitin ligase Nedd4-2 to inhibit ENaC by increasing ubiquitination and endocytosis of ENaC. Thus, enhanced expression of epithelial Na+ channels was detected in colon, airways, and kidney of AMPKα1−/− mice. Therefore, AMPKα1 is a physiologically important regulator of electrogenic Na+ absorption and may provide a novel pharmacological target for controlling epithelial Na+ transport.